The bacteriophage P1 cloning system allows the headful in vitro packaging of foreign DNA fragments as large as 95 kb in length. Sternberg, Proc. Natl. Acad. Sci. USA 87, 103-107 (1990) has shown that the P1 cloning system can generate 100,000 clones containing inserts per microgram of vector DNA. Large molecular weight clones are faithfully replicated in E. coli host strains and DNA from these clones can be easily isolated by standard molecular biological techniques. Thus, the P1 cloning system rivals Yeast Artificial Chromosomes (YAC) and cosmid cloning systems for the generation and characterization of genomic libraries.
Cosmid cloning vectors were designed by Bruning et al., Gene 4, 85-107 (1978) and Collins et al., Proc. Natl. Acad. Sci. USA, 75, 4242-4246 (1978), so that a bacteriophage lambda in vitro packaging reaction can encapsulate insert DNA up to 47 kb and infect E. coli at high efficiency. The cosmid vector plus insert DNA is cyclized in the E. coli bacterium at the lambda cos sites located on the vector. The same cos site is used in recognition by the lambda packaging apparatus for encapsulation of the vector-insert DNA into the lambda bacteriophage head. A major limitation of the cosmid cloning system is the relatively small size of the insert clone (47 kb). Many eukaryotic genes have been shown to be larger than 50 kb with some genes (e.g., dystrophin) up to 1000 kb. The small size of cosmid clones necessitates a labor intensive and "error-prone" procedure of multiple chromosome "walking" and "jumping" methodologies when isolating large genomic clones.
Another system for cloning large molecular weight DNA fragments is Yeast Artificial Chromosomes (YAC's) developed by Burke et al., Science 236, 806-812 (1987). YAC cloning enables DNA inserts up to 1000 kb to be propagated as minichromosomes in specific yeast strains YAC vectors contain a yeast replication origin, a centromere, and a set of telomeres. After ligation of insert DNA to the YAC vector, the DNA is introduced into yeast spheroplasts by direct DNA transformation. The major limitation of YAC cloning is the inefficiency of the transformation reaction (about 1000 clones per microgram of vector DNA) and the difficulty in characterization of YAC clones once they have been generated. The YAC clone represents a small proportion (less than 1%) of the total DNA in a yeast cell. This makes recovery, isolation, and analysis of any particular YAC clone burdensome.
The bacteriophage P1 cloning system complements both cosmid and YAC cloning in the construction of genomic libraries. A 50,000-member human DNA library has been generated in the P1 cloning system by Sternberg et al., The New Biol. 2, 151-162 (1990) which represents about a one times coverage of the human genome. The most recent P1 cloning vector (pNS582tet14Ad10) consists of a P1 pac site used for the initiation of headful packaging, two P1 lox sites which cyclize the P1 vector upon introduction in an E. coli host strain containing the P1 cre protein, a kanamycin gene for determining which E. coli cells contain a P1 plasmid, and a tetracycline gene for the cloning of insert DNA. The P1 cloning vector also contains a bacteriophage P1 plasmid replicon which maintains the P1 clone at a single copy per cell, and an IPTG inducible P1 lytic replicon for amplifying P1 clones in DNA isolation procedures. Another aspect of the cloning vector is a 10 kb "stuffer fragment" from adenovirus DNA which gives flexibility in the headful packaging reaction.
A model P1 cloning reaction consists of cutting the pNS582tet14Ad10 with the restriction enzymes ScaI and BamHI to generate 5 kb and 25 kb vector "arms". The digested vector DNA is then treated with calf intestine alkaline phospatase to inhibit self ligation of the vector. The vector arms are added to genomic DNA fragments that were previously digested with a BamHI-end compatible restriction enzyme (e.g. Sau3A). The two DNA's are then ligated and a portion of the ligation mixture is added to the first part of the two stage P1 in vitro packaging reaction. The first reaction consists of a cell extract prepared from P1 infected E. coli which is enriched for the P1 pac cleavage proteins. After pac cleavage, the DNA mixture is incubated in the stage II P1 in vitro packaging reaction which consists of a E. coli cell extract enriched for P1 virion capsids and tails. The phage encapsulated DNA is then infected into an E. coli host strain that contains the cre recombinase. A lox-lox site specific recombination reaction effectively cyclizes the P1 vector-insert clone which is maintained as a single copy extrachromosomal circular plasmid. To isolate DNA from a P1 clone, the cell containing the clone is grown in the presence of IPTG which induces the P1 lytic replicon. This induction increases the copy number of the P1 cone about 25 fold which gives enough DNA (about 1 microgram) from a 10 ml mini-alkaline lysis DNA isolation procedure for standard restriction mapping and size characterization procedures.
One problem encountered in the P1 cloning system is that a significant number of P1 vector molecules that contained no insert were present after a typical cloning experiment. These "no-insert" clones interfered with subsequent analysis of the cloning experiment in two ways. First, the number of clones to be screened when looking for a particular DNA insert was markedly increased due to the presence of "no-insert" containing clones. Secondly, upon subsequent growth of E. coli from a P1 cloning experiment, the bacteria that contained a "no-insert" vector generally grew much better than clones that contained large DNA inserts. Therefore, after a few rounds of growth the population of E. coli containing clones was greatly increased for "no-insert" vector clones.
To overcome the problems encountered in the previous versions of the P1 cloning system the pAd10-SacBII positive selection P1 cloning vector was developed. Many other positive selection based cloning systems have been developed for standard plasmid based recombinant DNA work. Henrich et. al., Gene 42, 345-349 (1986) demonstrated a positive selection vector based on the E gene (lysis protein) of bacteriophage .phi.X174. Kuhn et al , Gene 42, 253-263 (1986) developed a system which uses the EcoRI endonuclease. Burns et. al., Gene 27, 323-325 (1984) showed that positive selection can be generated in a system based on resistance to 5-fluorouracil. Other similar systems are listed in the Burns et al. article.
Another positive selection system used in DNA cloning is based on the sacB gene from Bacillus subtilis. This gene codes for the enzyme levansucrase (sucrose:2,6-.beta.-D-fructan 6-.beta.-D fructosyltransferase; EC 2.4.1.10) which catalyzes the transfructorylation of sucrose to various acceptor substrates resulting in the hydrolysis of sucrose and levan synthesis. Gay et al., J. Bacteriol. 164, 918-921 (1985) demonstrated that the production of levansucrase in E. coli is lethal in the presence of growth media containing 5% sucrose. Gay et al. have used this knowledge to develop a positive selection cloning system based on inactivating the B. subtilis sacB structural gene. This allows the growth of only those E. coli bacteria containing recombinant clones that have DNA inserts when grown in the presence of sucrose.
Tang et. al., Gene (in press) (1990), (U.S. patent application Ser. No. 07/376,474) have cloned the sacB gene from Bacillus amyloliouefaciens and shown extensive DNA sequence homology to the sacB gene from B. subtilis. When the sacB gene from B. amyloliouefaciens was cloned on a multicopy plasmid in E. coli, a lethal phenotype is observed when cells are grown in the presence of sucrose. This knowledge has inspired us to develop a novel P1 positive selection cloning vector, pAd10-SacBII.